At a time when concussions are a primary concern for athletes, a host of different companies and researchers are trying to improve their diagnosis. Over the years, there have been a number of tests for diagnosing and managing concussions, including the widely-used SCAT-3, and of course, the NFL’s concussion protocol. But all of these tests have the same limitation: they are fundamentally subjective.

Concussions, which are a form of traumatic brain injury (TBI), are primarily diagnosed through cognitive testing and the identification of clinical symptoms like dizziness, confusion and memory loss. While those symptoms can be valuable diagnostic tools, they are also nonspecific and can potentially go unreported by athletes who don’t want to get sidelined.

“In some of these sports, there’s almost a culture that you really want to be tough and you really don’t want to remove yourself from the game,” said Kevin Hrusovsky, CEO of the biotechnology company Quanterix. “But if there was an objective test, that could shift the culture of the game.”

One of Hrusovsky’s main goals with Quanterix is to create a concussion diagnosis instrument that can objectively test players on the sideline and quickly tell them whether or not they have a concussion. The appeal is obvious, but it’s a difficult task.

“I think the challenge lies in that this is a functional injury and not a structural injury,” said Teena Shetty, a neurologist at the Hospital for Special Surgery in New York. “Therefore a routine MRI is normal, but the patient is functionally impaired. That makes it more difficult to create a tool which can be administered both quickly and objectively on the sideline and have prognostic value.”

Nevertheless, many companies are striving to create such a tool. Shetty, who is also an unaffiliated neuro-trauma consultant for the New York Giants, can be seen narrating a recent NFL ad that features some emerging concussion technologies, many of which were created by companies that received funding through the league’s Head Health Initiative, a partnership with GE. Among those technologies is Quanterix’s Single Molecule Array, or Simoa, machine.

As the name suggests, the Simoa HD-1 Analyzeris a fully automated instrument that can detect single molecules in a selected fluid. Researchers can hunt for specific molecules by placing vials of blood or other fluids in the machine, at which point Simoa breaks down the fluid and mixes in antibodies and enzymes that will identify the target molecule. It’s the most advanced test of its kind, and Hrusovsky says it’s about a thousand times more sensitive than the traditional technology.

“Our technology allows scientists to see concentrations of proteins almost at the equivalence of a single grain of sand in 2,000 Olympic-sized swimming pools,” Hrusovsky said. “So that shows you just how dilute the concentration could be, and we could pick it up.”

Simoa is considerably advancing the study of concussions because researchers are using it to detect biomarkers — proteins whose concentrations can be indicative of TBI — that were previously undetectable. The most well-known of these concussion biomarkers is a protein called tau.

One of tau’s main functions is to stabilize the skeletal building blocks of axons — the fibers that carry signals throughout the brain. When the brain slams against the skull during a TBI, some of the tau gets dislodged from the axons and enters the surrounding fluid.

“Initially, these proteins get released into that cerebrospinal fluid,” Hrusovsky said. “But to get at that cerebrospinal fluid, you have to do a lumbar puncture, which is very painful and very invasive. It’s not something that you would ever do on a routine level. So the real interesting science was — does any of the tau that gets released in the cerebrospinal fluid find its way into blood?”

As it turns out, some of the tau in the cerebrospinal fluid does enter the bloodstream, crossing the semipermeable blood-brain barrier in the process. As a result, tau concentrations in the blood can become elevated following a TBI, which means that a simple drop of blood could potentially inform you of brain trauma. But this wasn’t possible before Quanterix created Simoa.

“There are a lot of questions about the physiology behind tau,” said Jessica Gill, a researcher at the National Institute of Nursing Research. “It has only really opened up recently because we weren’t able to detect it previously in peripheral blood since it’s in such low concentrations. So now that we have a new detection device, we can really look at it.”

Quanterix began selling the Simoa analyzer to researchers in 2013, and the company’s techniciansalso now run tests in their own lab for scientists who can’t afford the machine. As Quanterix’s technology became available, researchers soon recognized the potential of tau.

Henrik Zetterberg, a neuroscience professor at the University of Gothenburg in Sweden, performed some of the earliest research identifying tau as a blood-based biomarker for brain injury. Using Quanterix’s single-molecule assay, he published a 2013 study in Brain Injury, which showed that Olympic boxers had significantly elevated tau concentrations in plasma (the liquid component of blood) following a fight. He concluded that “repetitive minimal head injury in boxing may lead to axonal injuries that can be diagnosed with a blood test.”

In January 2017, Gill published a study in Neurology that took it a step further. After using Simoa to test the blood of 46 concussed athletes at the University of Rochester, she found that plasma tau can do more than just indicate brain trauma — it may also predict recovery time.

“The significant thing about this study is that, for the first time, we find that there’s a blood-based biomarker that also predicts return to play and that we can measure it within six hours and get some indication if that athlete can go back to play,” Gill said.

In the study, Gill tested each concussed athlete six hours, 24 hours, 72 hours, and seven days following the concussion. She found that among the concussed athletes, those who remained out of play for more than 10 days generally had higher plasma tau concentrations than those who were cleared to return to play sooner, especially when tested at the six-hour interval.

Gill’s conclusion — that plasma tau concentrations measured within six hours can help inform return-to-play decisions — is big. Many different problems can occur when a concussed athlete returns to play too soon, the deadliest of which is a rare phenomenon known as second-impact syndrome.

Second-impact syndrome occurs when an athlete returns to play following a head injury, only to suffer a second head injury that results in brain swelling, herniation, and sometimes death. There are only maybe a few dozen documented cases of the condition, but improving return-to-play decisions could go a long way toward preventing them in the future.

Despite the promise of Gill’s research, there are still many aspects of tau that require further study. “It’s exciting to see where it’s going, but it’s almost leading to more questions than answers,” she said. One of those questions is whether there are other factors, aside from concussions, that can raise tau levels in the blood. The study suggests that there are.

While plasma tau concentrations for concussed athletes spiked six hours after the concussion, they actually fell below the concentrations of the non-concussed control athletes at the 24-hour, 72-hour, and seven-day intervals. This suggests that plasma tau levels go back down within a day of a concussion and that other factors are raising the tau levels in the non-concussed athletes.

Gill speculates that one of those factors could be simple physical exertion, which previous studies have suggested could be linked to increased tau concentrations. Another factor could be sub-concussive blows, hits that don’t produce symptoms but may still cause brain injury. Whatever the reason, it’s clear that tau’s main prognostic value lies within the first hours following a concussion, when levels peak. Monitoring concussions beyond that time frame may require other biomarkers.

“While tau may be great for diagnosing a concussion, for management of concussions, it does really poorly,” said Jonathan Oliver, a kinesiology professor at Texas Christian University (TCU). “What if you don’t see the injury happen? What if we aren’t right there in the immediate time frame afterwards? What if somebody comes into a clinic with symptoms from a hit they took a few days ago? Tau’s not going to work.”

That’s why Oliver is studying a different biomarker called neurofilament light, or NF-L. NF-L is a protein chain that, like tau, is attached to axons in the brain and can fall off following a brain injury. However, compared to tau, NF-L levels can remain elevated in the blood for much longer. Oliver says that tau concentrations usually go back down after 12 hours, while NF-L concentrations can remain noticeably high for weeks.

Oliver also says that NF-L could be more valuable than tau when it comes to identifying sub-concussive hits. In 2015, he published a study, which was also co-authored by Zetterberg, in the Journal of Neurotrauma. In the study, Oliver used Simoa to periodically test the NF-L levels of TCU’s football players, excluding those who had been diagnosed with concussions. He found that the NF-L levels of the starters rose during points in the season when they likely endured the most impacts, implying that sub-concussive hits could be the cause.

While NF-L could play a big role in improving concussion management, Oliver admits that it’s only one piece of the puzzle. With so many groups performing concussion research, he thinks the future of concussion analysis could include NF-L, tau, and any number of other proteins.

“I believe there are different biomarkers that are useful, and it may come to a time where we find out that there’s a whole group of biomarkers for use,” Oliver said. “But right now, everybody’s just trying to differentiate themselves in the market.”

As an example, Oliver points to companies like Quanterix and Banyan Biomarkers (which, like Quanterix, received funding through the Head Health Initiative). While Quanterix holds the single molecule assay being used to detect tau and NF-L, Banyan Biomarkers owns the intellectual property rights to the testing of two other concussion biomarkers, UCH-L1 and GFAP. Other companies also hold exclusive rights to test other biomarkers.

While exclusive testing could impede the progress of concussion biomarkers, Quanterix has shown a willingness to work with other companies. In 2016, Quanterix established a license agreement with Banyan, allowing the company to incorporate the UCH-L1 and GFAP assays into its biomarker menu. By expanding the range of applications for Simoa, Hrusovsky hopes his company can leap over the hurdles that are slowing down the science.

The science, itself, is probably the biggest hurdle. Although concussion biomarkers look promising, researchers still don’t have baselines for most of them, which makes it difficult to determine “normal” and “elevated” levels across large populations. And if the goal is to create a definitive concussion test through blood, scientists need to directly connect the biomarker concentrations to brain injury, while weeding out other possible contributors.

“It’s just so complex,” Gill said. “You’re trying to get at brain activity through peripheral blood, and it’s just a very difficult problem to understand.”

Another obstacle is that the technology itself needs to be improved. While Quanterix’s technology is unmatched in its specificity, it still isn’t practical for quick sideline use.

“I think we have a long way to go,” Gill said. “The whole Simoa system is about as big as a soda vending machine, so we have to get that technology smaller, transportable. That’s what Quanterix works on.”

Although Simoa is very large, Quanterix is coming out with a new “desktop” version of the machine, which Hrusovsky says will be available in the fourth quarter of 2017 or the first quarter of 2018. Hrusovsky says the new version of the machine will be able to run the tests in about 30 minutes, compared to the original 45-minute analysis time of the current model. He also says the new model will be cheaper — dropping the $135,000 price of the current version to roughly $50,000-$55,000 — although it will require scientists to do more preparation work.

If all goes as planned, Hrusovsky hopes to eventually develop Simoa into a handheld device that can test for concussions in five minutes or less. That would be an invaluable tool for sports sidelines, and most of these researchers agreed that something like that could realistically exist within 10 years.

Still, achieving that goal is an uphill battle. Advancing the technology and miniaturizing the machine requires money, and Quanterix is still looking for investors. The company raised $46 million in 2016, but it will need to continue to receive funding, which Hrusovsky hopes will happen when he takes the company public later this year.

The goal, he says, is to “advance and accelerate the pace in which these technologies can be brought to commercial reality and everyday life.” Someday, he thinks Quanterix could even sell a version of the device at a CVS or Walgreens. But before that can happen, big strides need to be made, and many researchers think it will take some time.

“I don’t think it’s insurmountable, but we are still far from a single objective test which can be used in isolation,” Shetty said.

Shetty, herself, is approaching concussion research from a different angle. In 2014, she launched the second phase of a longitudinal neuroimaging study, which is being funded through the Head Health Initiative. In the study, she meets with concussed patients over the course of three or four encounters, performing multiple neurological assessments and imaging tests.

Although most concussions don’t show up on a typical MRI or CT scan, Shetty employs newer and more advanced imaging techniques like functional MRI (fMRI), diffusion tensor imaging (DTI), arterial spin labeling (ASL), susceptibility weighted imaging (SWI) and volumetric neuroimaging. The goal of the study is to identify biomarkers associated with microbleeds in the brain, changes in water movement in the brain, size changes for different brain regions, and metabolic changes in the brain cells. Together, those things can shed some light on how the brain changes in the days and weeks following a concussion.

BrainScope, another company that received grant money through the Head Health Initiative, is approaching brain injury from yet another direction. The company created BrainScope One (formerly known as the Ahead 300), an FDA-approved medical device that uses electroencephalography (EEG) technology to identify abnormal electrical activity in the brain following a TBI.

The device is an electrode headset that compares the patient’s brain electrical activity to a database of same-age controls and sends that EEG data to a smartphone app. The company’s CEO, Michael Singer, thinks that it provides a crucial contribution to the goal of objective concussion diagnosis.

“It is not meant to be the only indicator for whether somebody has a concussion,” Singer said. “It is meant to be a vital piece of objective information that can be deployed right then and there to be able to help the clinician. Our view is that it should be core to the armamentarium of things that a clinician will look at when they make clinical diagnoses.”

The combination of concussions and sub-concussive hits, repeated over time, is widely believed to cause chronic traumatic encephalopathy (CTE) — the neurodegenerative disease in which the brain slowly deteriorates from the inside. Symptoms of CTE don’t show up until years after the repetitive brain injury occurs, but they get worse as the disease progresses, eventually culminating in erratic behavior, depression, motor impairment, and dementia.

One of the pathological hallmarks of CTE involves a familiar protein — tau. When tau falls off the axons during a brain injury, it can become misfolded, resulting in a hyper-phosphorylated version of the protein called p-Tau. Eventually, after repeated hits, this p-Tau can aggregate into “neurofibrillary tangles.” The identification of these tangles has largely formed the basis for CTE diagnosis, but there’s one limitation: it can only be diagnosed by studying the brain tissue after death.

“The problem with CTE is that we’re pretty good at making a diagnosis by autopsy, but you can’t help many people out if you can only tell them they have a certain disease once they’re dead,” said Julian Bailes, co-director of the NorthShore University HealthSystem Neurological Institute and Director of the Department of Neurosurgery.

Bailes, a former team doctor for the Pittsburgh Steelers, played a pivotal role in the early findings related to CTE. In 2005, he reached out to Bennet Omalu — the doctor who originally discovered the disease in Steelers Hall-of-Famer Mike Webster — and together they challenged a resistant NFL to take action (their story is the subject of the 2015 film Concussion). Since then, hundreds of people have been diagnosed with CTE, including Hall of Fame linebacker Junior Seau, who took his own life in 2012.

Given the extreme nature of the disease and the expansive role of contact sports, diagnosing CTE in living people has become an ambitious goal for many scientists. A lot of researchers think positron emission tomography (PET) scans hold the key to identifying neurofibrillary tangles in living patients. Bailes, himself, has worked on a PET imaging probe called FDDNP to search for the tau deposits that make up these tangles.

Like most PET imaging probes, FDDNP is a radioactive tracer that is injected into the body intravenously. It enters the bloodstream and circulates throughout the body, eventually crossing the blood-brain barrier into the brain. If there are tau deposits in the brain, the tracer will bind to them and highlight them in the scan. The license for FDDNP is held by TauMark, a company owned by Omalu, UCLA researchers Jorge Barrio and Gary Small, and Robert Fitzsimmons — Webster’s former attorney.

At the UCLA Brain Research Institute, Barrio and Small have scanned hundreds of former football players and military veterans using FDDNP. In 2015, they published a study in Proceedings of the National Academy of Sciences in which they scanned 14 former football players and found patterns of protein deposition that appeared to be consistent with CTE. But FDDNP isn’t the only tau-binding tracer being researched.

In 2016, Mount Sinai neuroscience professors Dara Dickstein and Sam Gandy published a study in Translational Psychiatry, in which they used a tracer called T807 (also known as AV-1451 or Flortaucipir) to identify tau deposits in a retired NFL player who, based on his symptoms, likely had CTE. Dickstein says T807, which is licensed by Avid Radiopharmaceuticals, is better than FDDNP at identifying CTE-related patterns because it has a greater specificity for tau.

While T807’s affinity to tau is 25-times higher than its affinity to a protein-aggregate called amyloid, FDDNP binds strongly to both tau and amyloid. Dickstein says that can make it difficult for FDDNP to distinguish CTE from Alzheimer’s disease, since amyloid plaques are one of the pathological hallmarks of Alzheimer’s.

However, Bailes believes labelling amyloid is important, saying that up to 50 or 60 percent of CTE-positive patients can have amyloid deposits. He also says that FDDNP can still distinguish CTE from Alzheimer’s because the pattern of protein deposition, which includes the presence of tau deep in the folds of the brain, is distinctive.

“It’s not just what it’s labeling,” he said. “It’s the appearance, the pattern, which appears to be unique. It corresponds to what we see in autopsy, and it’s not seen in any other conditions.”

There are also other tau tracers being developed. Among them is the THK series of tracers, which were created at Tohoku University in Japan. Several researchers at Tohoku University published a study in The Journal of Nuclear Medicine in 2016, touting the latest tracer in the series, THK-5351, as one of the most sensitive tau tracers yet.

Another tau tracer, PBB3, was developed in Chiba, Japan, and it may have the ability to bind to diverse forms of tau in a wide variety of diseases. However, it has one drawback: it decays much more quickly than the others. While FDDNP, T807, and the THK series are all fluorine-18 tracers with a 110-minute half-life, PBB3 is a carbon-11 tracer with a half-life of only 20 minutes. That means PBB3 has to be synthesized on site, injected, and scanned very quickly, making it impractical for many labs.

Regardless of the tracer, the science behind these imaging probes is still fairly new. In 2015, the FDA sent TauMark a letter accusing the company of overstating the safety and effectiveness of FDDNP and forcing them to change the language used in its promotion. The FDA has still not approved FDDNP, or any tau tracer for that matter, and it could still be a while before that happens. Getting approval may require researchers to confirm their PET findings with a post-mortem CTE diagnosis. At the very least, it will require some longer-term studies.

“What would really be great is to do longitudinal studies to follow-up with our subjects,” Dickstein said. “Are we seeing any sort of changes in their behavior over time? Is it getting worse or getting better? Are we seeing changes in the structure of the brain? Are we seeing changes in the amount of brain matter? Are we seeing changes in the connectivity between brain regions? Are we seeing changes in the tau? Can it resolve itself over time? We don’t know. So having multiple scans from people over the span of a couple of years would be incredibly beneficial to the field and to the research.”

The other issue with PET scans is that they aren’t always easy for patients. They can take hours, they’re often expensive, and they expose patients to small amounts of radiation. For these reasons, PET scans will likely never be used for widespread screening, but that’s where Quanterix can help. If the company succeeds in creating a quick, handheld version of the Simoa machine, some scientists think it could be used to screen patients for CTE before a PET scan becomes necessary.

In a 2017 study in Alzheimer’s & Dementia, researchers used Simoa to perform blood tests on 96 former NFL players who had cognitive, behavioral, or mood complaints — symptoms that are suggestive of CTE. After determining their tau concentrations in blood, the researchers found that the players who were exposed to the most repetitive head injuries tended to have the highest tau levels.

“That was, to me, the most important and fascinating finding — that now, in middle-age, there was this direct relationship between their past exposure to hits and their current plasma tau,” said Robert Stern, the Director of Clinical Research at Boston University’s CTE Center and a co-author of the study.

That correlation was more than a little surprising. Considering that plasma tau concentrations usually go back down within a day of a concussion, it’s shocking to see them elevated in former players who haven’t taken hits in years. Stern suspects that the mechanism through which tau becomes elevated in CTE is much different than the way in which it becomes elevated in concussions.

“There is this tau that can be detected early on at the time of injury, but that probably just goes away under normal circumstances,” Stern said. “But in this case (CTE), you have hyper-phosphorylated tau in the cells. Then the cells begin to die and release that tau into the extracellular space, and then some of that can perhaps escape into the blood.”

In another study, Stern found that exosomes — vesicles that are released from cells and can be found in most bodily fluids, including blood and urine — may also be good biomarkers for CTE. After looking at the plasma exosomes of 78 former NFL players and 16 controls, he found that the player group generally had more tau in their exosomes and that it was highly correlated with cognitive issues.

In an effort to understand and develop all the potential angles for CTE diagnosis, Stern is also leading a seven-year longitudinal study that will put former NFL and college players through a battery of tests including a MRI, a lumbar puncture, two PET scans, blood and saliva collection, and a detailed neurological examination. The $16 million dollar project, which is being funded by the National Institute of Neurological Disorders and Stroke, is arguably the most comprehensive CTE study ever attempted. Stern thinks it will reap big rewards.

“All of that together will, in my mind, lead to the ability to diagnose CTE during life sometime in the next five years,” Stern said. His optimism wasn’t shared by most of the other researchers in this article, who almost unanimously agreed that a diagnostic tool for CTE in living patients is much further down the road.

Stern acknowledges that there are obstacles. For example, before a blood screening for CTE can truly be possible, the assays kits need to be refined. While Simoa has allowed scientists to detect the previously unseen tau, the technology is still not advanced enough to differentiate p-Tau, the hallmark of CTE, from other forms of tau in the blood. Quanterix is, however, developing a p-Tau assay kit that they expect to release in the third quarter of 2017.

But even when p-Tau assay kits do exist, blood tests will likely only ever be a small first step in CTE diagnosis. “A blood test is unlikely to be a be-all and end-all diagnostic for CTE because there are lots of diseases that can have the same types of problems in blood,” Stern said. And when it comes to the role of tau in CTE, the relationship is still poorly understood.

“In regards to CTE, I think the biology really needs to be figured out,” Dickstein said. “Does tau cause the disease or is it just a byproduct of what’s going on in the brain? That’s a big question, and I don’t think there’s enough out there to really know the answer to it.”

And then, even when a good CTE diagnostic for living patients does finally arrive, there are questions about how sports leagues will react to the news. Given the NFL’s historic mismanagement of CTE, including an infamous attempt to steer a research grant away from Stern, it wouldn’t be surprising to see the league reject the idea of a living CTE diagnosis altogether.

While an objective concussion test would diagnose an acute injury that usually results in a full recovery, a CTE test could indicate long-term and irreversible damage. If a CTE diagnostic finds evidence of the disease in, say, a large number of current NFL players, the league could experience a mass exodus.

A revelation like that could also change the nature of football itself, forcing rule changes that may make the game unrecognizable to fans. Hrusovsky isn’t too worried about that, but he acknowledges that improved safety measures would have some effects.

“I don’t think it’s ever going to lessen the importance of the game because athletics plays such an incredible role in society, but I think it could shift the way the game is played and the kind of protocols that might be in place to ensure the safety of the player,” he said.

For now, researchers can only guess how sports will be affected by developments in concussion and CTE diagnosis. All they can do is advance the science and see where it leads.